1. Field of the Invention
The present invention relates to a method for detecting fungal cells in clinical material.
Methods for detecting fungal cells in clinical material are of great interest because, especially in recent years, fungal species have acquired considerable importance as significant nosocomial pathogens, in particular for immunosuppressed patients. If fungal infections are not recognized in time in such patients, they propagate in the patient's body and result in a high mortality rate. Treatment success can be improved only by timely diagnosis.
2. Related Prior Art
The standard methods known for the detection of fungal infections, which are based on the culturing of isolated fungi, are complex and time-consuming. On the other hand, fungal infections can be detected sensitively and promptly using new techniques based on molecular biology methods. These methods require, however, the ability to efficiently extract fungus-specific nucleic acids from clinical material. In order to identify, from these extracted nucleic acids, the fungal species responsible for the infection, specific sequences of different pathogenic fungal species must be known. This requires differentiating among the various fungal species, since the particular therapy depends on the individual fungal infection.
With this background, a method has been developed with which fungal DNA can be extracted from patient material, the fungal DNA can be analyzed, and various fungal species can be identified on the basis of the extracted DNA. This method is described in DE Patent Application 195 30 336.9 as follows:
First the fungal DNA is extracted from the patient's whole blood. This is done by first isolating fungal cells from blood cells. Then the fungal cells are lysed and their DNA is purified from the lysate. Fungus-specific DNA segments from the fungal cell DNA thus obtained is amplified in a polymerase chain reaction (PCR). This polymerase chain reaction is performed with two primers which amplify a region, comprising approximately 500 nucleotides, from the gene for 18ssu rRNA. The primers are selected so as to amplify only the corresponding gene region from fungi, but not gene regions from other organisms, for example the patient's own body cells. The sequences of these primers are listed in the attached Sequence Listing as SEQ ID No. 1 and 2.
A DNA fragment is thus amplified in the polymerase chain reaction only if a fungal infection is present. Considered in and of itself, the PCR thus serves to detect the existence of an infection with pathogenic fungi.
In order then to differentiate among different fungal species, the amplified 500-base-pair fragment is hybridized with one or more of a total of six probes. Each probe is specific for one fungal species. According to DE Patent Application 195 30 336.9, specific probes are indicated for a total of five Candida species and the genus Aspergillus. The probes are also listed in the Sequence Listing attached hereto, as SEQ ID No. 3 through 8.
The fungal genera Candida and Aspergillus are those by far most often involved in nosocomial infections. Less-common fungal species, for which so far no detection methods are available, are nevertheless also gaining in clinical significance.